mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Yeast Three Hybrid (Y3H) System
Saccharomyces cerevisiae is a species of budding yeast. It has a relatively short life cycle (100 minutes when nutrients are abundant) and is easy and inexpensive to maintain and grow 2. Besides, yeast cell is easy for genetic manipulation (like transformation). These advantages make S. cerevisiae an ideal model organism in eukaryotic genetics 2. Yeast-two-hybridization is a widely used approach to detect and screen for protein-protein interaction. The majority of small molecule drugs act on one or multiple protein targets for the therapeutic function. Yeast-three-hybridization is a derivative of yeast-two-hybridization to detect and screen for the interaction between small molecule drugs and their protein targets 1. The principle of yeast-three-hybridization 1. Components of a typical yeast-three-hybrid system Similar to yeast-two-hybrid, the yeast-three-hybrid system also uses a “bait” fusion protein (protein-of-interest X attached to the DNA binding domain of a transcription activator) and a prey protein fusion (protein-of-interest Y attached to the transcription activation domain of the same transcription activator) 1. The major distinguishing characteristic of yeast-three-hybrid is the attachment of an “anchor” molecule and a “linker” molecule attached to the bait fusion protein, thus allowing the attachment of the small molecule drug to the “bait protein scaffold” 1. The anchor molecule is normally a compound that has high affinity to the bait protein it attaches so that a bait scaffold can be formed for the drug molecule to attach. The long linker molecule provides a flexible bridge between the anchor and the drug molecule so that the drug-target interaction is not affected by conformation factors 1. 2. Screen for drug-target interaction First the yeast is transformed with the gene encoding the bait fusion protein. The yeast expressing the bait scaffold is then loaded with the small molecule drug. After that, the yeast will is transformed with a cDNA library encoding the target protein (“prey”) fusion 1. Thus the prey protein fusion will be expressed and the drug will interact with the target protein. The interaction between the drug and the target will be identified by the transcription of the reporter gene (e.g. LacZ, antibiotic resistance) 1. The plasmid will be isolated and sequenced from the yeast cells with positive results and the target will be identified. This drug-target interaction will be further verified by in vitro assays (e.g. immunoprecipitation) 1. Potential pitfalls and solutions False-positives and false-negative results are associated with yeast three hybrids. False positive refers to the hypothetical drug-target interactions that are detected by yeast-three-hybrid but cannot be replicated by other approaches. False negative refers to the drug-target interactions that are predicted by other approaches but cannot be detected by yeast-three-hybrid 1. For false positive results, a control “drug” molecule can be introduced into the assay system to verify that all the drug-target interactions identified are specific between the drug of interest and the protein target. An alternative approach is to perform a pre-screening by expressing the cDNA library of prey fusion protein (protein target linked to the transcription activation domain) without loading the drug molecule. The pre-screening can indicate whether there are nonspecific interactions between the bait scaffold and the target protein 1. One cause of false negative is that yeast-three-hybrid takes place in the nucleus of the yeast cell, because the players of interaction are fused with transcription factors. So if the target protein is a dedicated membrane protein, it will be difficult for the target protein to get inside of the nucleus. Another source of the false negative is the inefficient load of drug molecule, due to the solubility of the drug or the membrane permeability of the yeast cell used. The problem can be bypassed by utilizing permeability altered yeast strains or the drug efflux pump deficient strains 1. References 1. Rezwan, M. & Auerbach, D. Yeast “N”-hybrid systems for protein-protein and drug-protein interaction discovery. Methods 57, 423–9 (2012). 2.http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae